The present invention relates to novel binding peptides and, more particularly, to small peptides that bind to the thrombospondin 1 receptor.
The interaction of cells with extracellular matrix (ECM) molecules is a complex process from which cells derive a wealth of information about their environment. This information is processed in a number of ways that ultimately affect cell motility, shape, proliferation and gene expression (Hynes, 1992). ECM macromolecules like fibronectin, laminin, vitronectin, and collagen have been shown to mediate cell adhesion, a process that includes cell attachment and spreading. Like these proteins, thrombospondin 1 (TS1) promotes adhesion of a number of normal and transformed cell types (Frazier, 1991; Roberts et al., 1987), a function which underlies many effects that TS1 exerts in several biologically complex systems. These effects include stabilizing platelet aggregation (Leung et al., 1984; Dixit et al., 1985), regulating cell growth (Majack et al., 1988; Good et al., 1990), specifying the differentiation phenotype of certain cells (Castle et al., 1991), wound healing (Raugi et al., 1987) and the migration of tumor cells (Tuszynski et al., 1987) and PMNs (Mansfield et al., 1990). A good example of the regulation of several aspects of cellular behavior by TS1 is the inhibition of angiogenesis in vivo and of endothelial cell migration and proliferation in vitro (Good et al, 1990; Taraboletti et al., 1990).
There are at least 4 TS isogenes, TS1, 2 and 3 (Bornstein et al., 1991; LaBell et al., 1992; Laherty et al., 1992; Vos et al., 1992) and cartilage oligomeric matrix protein or COMP (Oldberg et al., 1992) whose products are related, but decidedly different. Of these, platelet TS (which is pure TS1) is the best characterized, and serves as a prototype for this growing family. Distinct activities can be assigned to certain domains. For example, the amino-terminal domain of TS1 induces spreading of G361 cells while the COOH-terminal cell binding domain (CBD) of TS1 promotes haptotaxis and attachment of these cells (Taraboletti et al., 1987; Roberts et al., 1985). TS1 contains at least four domains that support cell attachment: the amino-terminal heparin-binding domain (Murphy-Ullrich et al., 1987), the type 1 repeats of about 60 amino acid residues containing a common subhexapeptide sequence (Prater et al., 1991), the RGD sequence in the last of the type 3 calcium binding repeats (Lawler et al., 1988) and the COOH-terminal ca. 220 residues termed the xe2x80x9ccell-bindingxe2x80x9d domain (CBD, Kosfeld et al., 1991). Monoclonal antibody (MAb) called C6.7 which binds to this CBD and blocks its interaction with cellular receptors was previously described (Dixit et al., 1985). Using this MAb it has been shown that the CBD is essential for binding of TS1 to platelets (Dixit et al., 1985), many transformed cells (Varani et al., 1986) and to human melanoma cells (Taraboletti et al., 1987). The CBD of TS1 (rCBD) exclusive of the upstream RGD sequence has been expressed in bacteria and its attachment activity for human melanoma cells has been demonstrated (Kosfeld et al., 1991).
In accordance with the present invention, novel small synthetic peptides are provided that bind to the thrombospondin 1 (TS1) receptor. These peptides preferably have 5-13 amino acid residues which share the tripeptide Val-Val-Met and have the following sequences:
RFYVVMWKQVTQS [SEQ ID NO:1] and fragments thereof containing the minimal sequence RFYVVM [SEQ ID NO:3], and
FIRVVMYEGKK [SEQ ID NO:4] and fragments thereof containing the minimal sequence IRVVM [SEQ ID NO:5].
The novel VVM-containing peptides of the invention are illustrated by five preferred pep tides in which the sequences are converted to the three-letter abbreviations and designated herein and in the Sequence Listing of the accompanying Diskette as f follows:
The foregoing 5 illustrative peptides are also designated herein for structural purposes as 4N1, 4N1-1, 4N1-2, 7N3 and 7N3-1, respectively.
The novel binding peptides of this invention are contained in 2 non-overlapping 30-residue synthetic peptides, designated C4 and C7, respectively, of the thrombospondin 1 (TS1) COOH-terminal cell binding domain (CBD). These novel peptides retain the binding activity of the parent 30-mer peptides and faithfully reflect the binding activity of CBD.
These results were unexpected in view of the fact that, by way of distinction, the following closely related peptides were inactive in said binding:
Gly-Arg-Val-Val-Met [SEQ ID NO:6],
Ile-Glu-Val-Val-Met [SEQ ID NO:7] and
Ile-Arg-Val-Val-Gly [SEQ ID NO:8].